Sphingoid compounds for prophylaxis and/or therapy of coronaviridae infection

ABSTRACT

Provided are processes for the prevention or treatment of infection by SARS-CoV-2. Some aspects provide administration of a sphingoid compound, optionally sphingosine, an active ingredient that activates generation of a sphingoid base, optionally of sphingosine; or an active agent that inhibits the degradation of a sphingoid base, optionally of sphingosine, to a subject such as a human. By increasing the local concentration of sphingosine in a subject or an area of a subject to which the composition is administered such as the airways, nose or interior of the nose or portion thereof, the ability or SARS-CoV-2 to infect the subject or cells thereof is reduced thereby treating of preventing infection by the virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application depends from and claims priority to U.S. ProvisionalApplication No. 63/052,520 filed Jul. 16, 2020, the entire contents ofwhich are incorporated herein by reference.

FIELD

This disclosure relates generally to treatments or prophylactics forviral infection in an organism, and more particularly infection by avirus of the Coronaviridae family.

BACKGROUND

Viral infections are the result of passive or active penetration of avirus and/or viral genome (ribonucleic acid or deoxyribonucleic acid)into a cell of an organism. Viral infection may also lead to subsequentpropagation of the virus within the organism. The process of viralpenetration into a cell requires overcoming the cell membrane typically.Some viral infection occurs by an endocytotic mechanism whereby aninvagination of the cell membrane is created to form a vesicle(endosome) resulting in the passive uptake of the virus into the cell.Within the cell, the virus escapes from the endosome by fusion of theouter viral membrane with the membrane of the endosome. Alternatively,the virus actively injects the viral genome into the cytoplasm of thecell without itself entering into the cell. Both mechanisms ultimatelylead to the entry of viral genome into the cytoplasm of the cell.

Viral penetration into the cell is usually followed by a propagation ofthe virus within the cell and subsequent release of the propagated virusfrom the cell. The released virus is subsequently capable of infectingfurther cells. A viral infection may be followed by a viral infectiousdisease, which manifests itself in terms of symptoms based on the viralinfection.

Many attempts have been made to address viral infections by targetingparticular components of the viral life cycle. Drugs that target viralreplication within the cell represent a key inhibitory target mechanismin the field. Alternatively, some attempts have been made to target themechanisms by which viruses enter a cell. For example, WO 2004/017949attempts to prevent viral entry into a cell by disrupting ceramide-richlipid rafts on the surface of cells. The reference attempts to useinhibitors of acid sphingomyelinase and/or inhibitors of ceramide orphosphorylcholine, products of the reaction catalyzed by this enzyme toalter the presence and characteristic of lipid rafts on the membranesurface.

Each of the viral targeting mechanisms of the past suffer significantdrawbacks such as viral mutation leading to escape and reducedeffectiveness of compositions, unwanted systematic or other sideeffects, the ability to target only a single virus type, or low success.As such, new compositions and mechanisms are needed for prophylaxis ortreatment of viral infection.

SUMMARY

This disclosure provides new compositions and methods for theprophylaxis or treatment of viral infection in a subject. Thecompositions may be used to target infection by a single virus type,illustratively a virus of the Coronaviridae family, and morespecifically SARS-CoV-2. As such, provided are sphingoid compositionsfor use in the prophylaxis and/or therapy of a viral infection bySARS-CoV-2.

BRIEF DESCRIPTION OF THE DRAWINGS

The aspects set forth in the drawings are illustrative and exemplary innature and not intended to limit the subject matter defined by theclaims. The following detailed description of the illustrative aspectscan be understood when read in conjunction with the following drawings.

FIG. 1 illustrates a schematic of the natural metabolism ofsphingomyelin (SM) illustrating the synthesis and breakdown ofsphingosine (SPH).

FIG. 2 illustrates the amount of lymphocytic choriomeningitis virus(LCMV) particles in the supernatant of Raw264.7 cells were treated with250 μM D-erythro-sphingosine for 10 minutes and then infected with theLCMV where the Y-axis: Infectious LCMV particles in the supernatant(PFU/ml, logarithmic scale), the X-axis: is time points of themeasurements (in hours after infection); Black bars are control; Whitebars are sphingosine-treated illustrating that treatment withsphingosine reduced propagation of LCMV.

FIG. 3 illustrates the amount of LCMV particles in the supernatant ofRaw264.7 cells were treated with ceramidase (250 mUnits/ml) for 10minutes and then infected with the LCMV (MOI: 0.01) where the Y-axis:Infectious LCMV particles in the supernatant (PFU/ml, logarithmicscale), the X-axis: is time points of the measurements (in hours afterinfection); Black bars are control; White bars are ceramidase-treatedillustrating that treatment with ceramidase reduced propagation of LCMV.

FIG. 4 illustrates the amount of LCMV particles in the supernatant ofRaw264.7 cells were treated with sphingomyelinase (6.5 U/ml) for 10minutes and then infected with the LCMV (MOI: 0.01) where the Y-axis:Infectious LCMV particles in the supernatant (PFU/ml, logarithmicscale), the X-axis: is time points of the measurements (in hours afterinfection); Black bars are control; White bars are ceramidase-treatedillustrating that treatment with ceramidase reduced propagation of LCMV.

FIG. 5 illustrates Hela cells treated with 10 micromolar (μM) and 100 μMsphingosine kinase inhibitor SKII and infected with the LCMVillustrating that inhibition of sphingosine kinase inhibits spreading ofthe virus.

FIG. 6 illustrates macrophages from wild-type (WT) mice andceramidase-deficient mice (Asah1^(−/−)) and then infected with herpessimplex virus (HSV-1) (MOI 5) illustrating that ceramidase deficiencypromotes the spreading of the virus.

FIG. 7 illustrates propagation of herpes simplex virus (HSV-1) in WTmice and ceramidase-deficient mice (Asah1^(−/−)) demonstrating thatceramidase deficiency promotes the spreading of the virus in the liverof HSV-1 infected animals.

FIG. 8 illustrates survival of control mice and inducibleceramidase-deficient mice treated with tamoxifen in order to switch offthe ceramidase demonstrating that ceramidase deficiency reduces thesurvival of the HSV-1 infected animals wherein Y-axis: Survival inpercent; X-axis: Time after infection in days; and Treatment groups:Black squares: infected control animals; Black circles: uninfectedcontrol animals; White squares: infected ceramidase-deficient animals;White circles: uninfected ceramidase-deficient animals; p=0.0004.

FIG. 9 illustrates propagation of LCMV in WT mice andceramidase-deficient mice (Asah1^(−/−)) as measured in the liverdemonstrating that ceramidase deficiency promotes the spreading of thevirus in the liver of infected animals.

FIG. 10 illustrates propagation of lymphocytic choriomeningitis virus(LCMV) in WT mice (black bars), ceramidase-deficient mice (Asah1^(−/−))(white bars) and interferon-alpha receptor-deficient mice (Ifnar^(−/−))(gray bars) as measured in the blood (A), bone marrow (B), liver (C),lung (D), kidney (E), heart (F) and where Y-axis: Infectious LCMVparticles in the organs measured (PFU/organ or PFU/ml of blood,logarithmic scale) demonstrating that ceramidase inhibits thepropagation of LCMV in all the organs tested.

FIG. 11 illustrates survival of control mice and inducibleceramidase-deficient mice treated with tamoxifen in order to switch offthe ceramidase demonstrating that ceramidase deficiency reduces thesurvival of the LCMV infected animals wherein Y-axis: Survival inpercent; X-axis: Time after infection in days; and Treatment groups:Black squares: infected control animals; White squares: infectedceramidase-deficient animals; White circles: uninfectedceramidase-deficient animals; (p<0.0001).

FIG. 12 illustrates Vero cells treated with 0, 10 and 50 mU/200microliters (μl) ceramidase for 10 minutes and then infected with thevesicular stomatitis virus (VSV) demonstrating that the ceramidaseinhibits the propagation of VSV where Y-axis: Number of resultantplaques per 24 wells and X-axis: Ceramidase concentration (mU/ml).

FIG. 13 illustrates Vero cells treated with 0, 250 and 1250 mU/mlsphingomyelinase for 10 minutes and then infected with the vesicularstomatitis virus (VSV) demonstrating that sphingomyelinase inhibits thepropagation of VSV where Y-axis: Number of resultant plaques per 24wells and X-axis: sphingomyelinase concentration (mU/200 μl).

FIG. 14 illustrates survival of control mice and inducibleceramidase-deficient mice treated with tamoxifen in order to switch offthe ceramidase. The animals were infected intravenously with thevesicular stomatitis virus (VSV) where the results demonstrateceramidase deficiency reduces the survival of the infected animals andwherein Y-axis: Survival in percent; X-axis: Time after infection indays; and Treatment groups: Black squares: infected control animals;White squares: infected ceramidase-deficient animals; p=0.0041.

FIG. 15 illustrates the number of virus particles after 3 days in WTmice and ceramidase-deficient mice (Asah1^(−/−)) infected intranasallywith the influenza A virus demonstrating that ceramidase prevents thepropagation of IAV wherein Y-axis: Infectious IAV particles in the lung(PFU/lung, logarithmic scale) and Treatment groups: Black bars: infectedcontrol animals; White bars: infected ceramidase-deficient animals.

FIG. 16 illustrates percentage of infected erythroid progenitor cellsafter 6 days following infection of ceramidase-deficient mice(Asah1^(−/−)) were infected intravenously with the Friend virus (FV)demonstrating that ceramidase prevents the propagation of FV whereinY-axis: Infected erythroid progenitor cells in percent and treatmentgroups: Black bars: infected control animals; White bars: infectedceramidase-deficient animals.

FIG. 17 illustrates propagation of HIV-P24 in human T cells as controlor treated with 50 μM of the sphingosine kinase inhibitor MP A08demonstrating that sphingosine kinase inhibition prevents thepropagation of HIV wherein Y-axis: Amount of P24 (HIV antigen) in ng/mland treatment groups: Black bars: untreated; White bars: inhibitor.

FIG. 18 illustrates binding of lymphocytic choriomeningitis virus tocontrol liposomes, and liposomes enriched with ceramide or sphingosinedemonstrating that sphingosine-containing liposomes can bind a virusrapidly wherein Y-axis: Virus binding (mean fluorescence intensity) andTreatment groups: Black bars: control liposomes; Grey bars: ceramideliposomes; White bars: sphingosine liposomes.

FIG. 19 illustrates micrographs of the liposomes of FIG. 18 whereinwhite are LCMV binding to the liposomes. ø: control liposomes; CER:ceramide liposomes; SPH: sphingosine liposomes.

FIG. 20 illustrates the prevention of RSV2 and RSV14 infection in Helacells treated with sphingosine relative to control and independent ofwhether sphingosine was added prior to or following infection.

FIG. 21 illustrates that application of sphingosine to the nose of miceresults in a dose dependent increase in the local sphingosineconcentration in the nasal mucosa and that this increase was notappreciably removed by washing.

FIG. 22 illustrates application of sphingosine into the nose of miceresults in no toxic side effects.

FIG. 23 illustrates that administration of prophylactic orpost-infection nasal administration of sphingosine to wild-type miceprevents infection by RSV2.

FIG. 24 illustrates that sphingosine-coupled agarose beads efficientlybind RV2 or RV14 wherein the Y-axis is the percentage of dead Hela cellsfollowing incubation with the supernatant of sphingosine-coupled agarosebeads with virus.

FIG. 25 illustrates that RV1b-coupled agarose beads efficiently bindsphingosine wherein the Y-axis is the remaining sphingosine in thesupernatant in μM.

FIG. 26 illustrates pH affects on the binding of sphingosine to RV2 orRV14.

FIG. 27 illustrates that sphingosine prevents infection of freshlyisolated human nasal epithelial cells by both RV2 and RV14.

FIG. 28 illustrates that sphingosine prevents infection of Vero cells bySARS-CoV-2 without negative effects on the cells.

FIG. 29 illustrates that sphingosine prevents infection of freshlyisolated human nasal endothelial cells by SARS-CoV-2 without negativeeffects on the cells.

FIG. 30 illustrates that sphingosine binds the ACE2 receptor

FIG. 31 illustrates that sphingosine prevents binding of SARS-CoV-2spike protein to the ACE2 receptor.

DETAILED DISCLOSURE

Provided in this disclosure are compounds and methods for the treatmentor prophylaxis of viral infection in a subject such as a mammal. On thebasis of cell and animal experiments, the inventors were able todemonstrate that, sphingoid compounds, such as sphingosine, and/oractive ingredients which influence the degradation and/or the generationof a sphingoid compound, optionally of sphingosine, in vivo, such asceramidase agonists and/or sphingosine kinase inhibitors, can be usedfor a prophylaxis and/or therapy of viral infections and/or viralinfectious diseases. For instance, the inventors were able to show inparticular that it is possible to prevent a viral infection of multipledifferent virus types including members of the Coronaviridae family suchas SARS-CoV-2 by means of an increase in the in vivo concentration ofsphingosine or to successfully control a viral infection that hasalready occurred and/or a viral infectious disease which is alreadymanifested by means of an increase in the in vivo concentration ofsphingosine. Furthermore, the inventors were able to show that a viralinfection and/or viral infectious disease is prevented by an increasedin vivo concentration of sphingosine relative to standard baseline. Assuch, provided are robust, rapid, and efficient methods of preventing orreducing viral infection of a subject and/or to the cells of a subject.

Processes as provided herein according to some aspects includeadministering to a subject a sphingoid compound, optionally a sphingoidbase, prior to, concomitant with, or following the subject beingcontacted by a virus.

As used herein, a “subject” is a human, a non-human animal, such as, forexample: a non-human mammal optionally a horse, cow, pig, dog, or cat; abird optionally a chicken, turkey, goose, or other; a fish; a reptile;an amphibian; a mollusc; an insect or a spider; a cell; or a plant.

As used in this disclosure, the term “sphingoid base” can mean a singletype of sphingoid base (singular) or a plurality of different, i.e. twoor more different, sphingoid bases, optionally a mixture of differentsphingoid bases. A sphingoid base includes salts thereof and/or hydratesthereof and/or solvates thereof and/or polymorphs thereof and/or opticalisomers, optionally enantiomers and/or diastereomers and/or epimers,thereof and/or mixtures thereof.

As used in this disclosure, the term “active ingredient” can mean asingle type of active ingredient (singular) or a plurality of different,i.e. two or more different, active ingredients, optionally a mixture ofdifferent active ingredients.

As used in this disclosure, the term “prophylaxis” is to be understoodto mean a preventive measure suitable for preventing a viral infectionand/or viral infectious disease. The preventive measure can optionallybe administering a sphingoid based as provided herein to a subject priorto exposure to a virus or reexposure to a virus. Optionally, thepreventative measure is a vaccination, and this will be discussed indetail below.

As used in this disclosure, the term “therapy” is to be understood tomean the treatment of a viral infection and/or viral infectious disease.The treatment can be either a causal treatment (so-called causaltherapy), i.e. a treatment aiming at the elimination of the cause of thedisease, or a symptomatic treatment (so-called symptomatic therapy),i.e. a treatment aiming at the elimination of symptoms. Therapy may beamelioration of one or more symptoms of a viral infection or viralinfection disease. Therapy may be the reduction of viral load in a cell,tissue or organism relative to control or prior to administration.

As used in this disclosure, the term “viral infection” is to beunderstood to mean viruses actively or passively penetrating into,remaining in and subsequently propagating in an organism, such as, forexample, a human, an animal or a plant.

As used in this disclosure, the term “viral infectious disease” is to beunderstood to mean a disease brought about by viruses, illustratively inhumans, animals or plants.

As used in this disclosure, the term “disinfection” is to be understoodto mean a hygiene measure which serves to bring dead or living materialinto a state where infection by said material is no longer present. Asused in this disclosure, the expression “disinfection” is preferably tobe understood to mean a hygiene measure which serves to kill orinactivate pathogens, preferably viruses, or to reduce their number onor in an object or on/in a biological surface.

Therefore, as used in this disclosure, the term “disinfection” can meanin particular an areal disinfection, i.e. a disinfection of areas,optionally surfaces, or a room-air disinfection, i.e. a disinfection ofroom air. For example, the disinfection can be a disinfection of theroom air of an intensive-care unit, an isolation unit, an operatingtheatre, a laboratory, an airport or an animal husbandry facility.

Provided in this disclosure are composition and processes of using oneor more of the composition to treat or prevent infection of a subject bya one or more viruses. A process includes administering to a subject inneed of prevention or treatment of viral infection a composition thatincludes an active ingredient. In some aspects, an active ingredient isor includes a sphingoid compound. Optionally, an active ingredient asprovided herein may be used as an active ingredient in a compositionalone or with other active or inactive molecules. Optionally, the activeingredient influences, in particular activates or inhibits, thedegradation or the generation of a sphingoid base, optionally ofsphingosine, in vivo for application or use in the prophylaxis and/ortherapy of a viral infection and/or viral infectious disease, in asubject as provided herein or for application or use in disinfection.

In some aspects, an active ingredient is or includes a sphingoidcompound. A sphingoid compound is optionally a sphingoid base, or otherform of a sphingoid compound suitable for administration to a subject.Optionally, a sphingoid compound is a sphingoid base optionally havingthe structure of formula I.

whereR¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), where R^(a) isa hydrogen atom, an alkyl radical optionally with a substituent ofamine, a quaternary ammonium optionally including 3 hydrogens, or asugar radical,R⁴ and R⁵ independently of one another mean a hydrogen atom, oxygenatom, a hydroxyl group, an amine, or a quaternary ammonium optionallyincluding 3 hydrogens,R⁶ is an alkyl radical optionally comprising one or more substituents ofN, a quaternary ammonium optionally including 3 hydrogens, or

where R² and R³ independently of one another mean a hydrogen atom or analkyl radical,n means an integer of from 2 to 50, optionally 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16, and

means a double bond or single bond,and/or salts thereof and/or hydrates thereof and/or solvates thereofand/or polymorphs thereof and/or optical isomers, optionally enantiomersand/or diastereomers and/or epimers, thereof and/or mixtures thereof.

In some aspects, a sphingoid compound as provided herein may be orinclude the following formula I-a

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical, a quaternary ammoniumoptionally including 3 hydrogens, or a sugar radical,R² and R³ independently of one another mean a hydrogen atom or an alkylradical,R⁴ and R⁵ independently of one another mean a hydrogen atom, a hydroxylgroup, or a quaternary ammonium optionally including 3 hydrogens,n means an integer of from 2 to 50, optionally 10, 11, 12, 13, 14, 15 or16, and

means a double bond or single bond,and/or salts thereof and/or hydrates thereof and/or solvates thereofand/or polymorphs thereof and/or optical isomers, optionally enantiomersand/or diastereomers and/or epimers, thereof and/or mixtures thereof.The compound of formula I may be used in the prophylaxis and/or therapyof a viral infection and/or viral infectious disease in a subject asprovided herein.

In some aspects, the sphingoid base according to the present disclosurecan have or include the following formula I-b:

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical or a sugar radical, or aquaternary ammonium optionally including 3 hydrogens,R² and R³ independently of one another mean a hydrogen atom or an alkylradical,R⁴ and R⁵ independently of one another mean a hydrogen atom, a hydroxylgroup, or a quaternary ammonium optionally including 3 hydrogens, andn means an integer of from 2 to 50, optionally 10, 11, 12, 13, 14, 15 or16.

Alternatively, the sphingoid base as provided herein can have or includethe following formula I-c:

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical or a sugar radical,R² and R³ independently of one another mean a hydrogen atom or an alkylradical,R⁴ and R⁵ independently of one another mean a hydrogen atom, oxygenatom, or a hydroxyl group, andn means an integer of from 2 to 50, optionally 10, 11, 12, 13, 14, 15 or16.

The sphingoid base according to formula I can in principle be anaturally occurring sphingoid base or a non-naturally occurringsphingoid base.

Furthermore, the sphingoid base as provided herein can have chiralcenters, optionally chiral carbon atoms, having the absoluteconfiguration R or S or R,S or d,D or l,L or d,l or D,L.

Furthermore, the sphingoid base can be a O-threo isomer, L-threo isomeror an L-erythro isomer of the sphingoid base according to formula I,I-a, I-b, or I-c.

Furthermore, R^(a) in the formula I can be a linear (i.e. unbranched),or branched alkyl radical, optionally having one to four carbon atoms.As an illustrative non-limiting example, the alkyl radical can be amethyl radical, an ethyl radical, a propyl radical, an isopropylradical, a butyl radical, a sec-butyl radical or a butyl radical.

In some aspects, R^(a) in the formula I is a hydrogen atom. In otherwords, R¹ in the formula I, I-a, I-b, or I-c optionally is CH₂OH.

The sphingoid base according to formula I can include at least 2 carbonatoms, optionally at least 6 carbon atoms. In some aspects, thesphingoid base can include 18 carbon atoms to 50 carbon atoms.Optionally, the sphingoid base according to formula I, I-a, I-b, or I-cincludes 18 carbon atoms.

Alternatively, R¹ in the formula I can mean a hydrogen atom (H), amethyl radical (CH₃), CH₂OCH₃, CH₂—O-galactosyl or CH₂—O-glucosyl.

As already mentioned, R^(a) in the formula I can also mean a sugarradical. The sugar radical can, for example, mean a monosaccharideradical, optionally a pentose or hexose radical. Optionally, the sugarradical is present in ring form, optionally as furanose or pyranose.

The sugar radical can, for example, be an arabinose radical, riboseradical, xylose radical, allulose radical, aldotriose radical, fructoseradical, galactose radical, glucose radical, gulose radical, inositolradical, mannose radical, or sorbose radical.

Optionally, R² and R³ in the formula I both mean a hydrogen atom.

Optionally, R⁴ in the formula I is a hydroxyl group and R⁵ in theformula I is a hydrogen atom.

In some aspects, the sphingoid base has the following formula II:

wheren means an integer of from 2 to 50, optionally 10, 11, 12, 13, 14, 15 or16, and

means a double bond or single bond.

Accordingly, the sphingoid base can have the following formula II-a₀:

wheren means an integer of from 2 to 50, optionally 10, 11, 12, 13, 14, 15 or16.

Alternatively, the sphingoid base can have the following formula II-a₀*:

wheren means an integer of from 2 to 50, optionally 10, 11, 12, 13, 14, 15 or16.

In some aspects, the sphingoid base according to formula I issphingosine, dihydrosphingosine, phytosphingosine,dehydrophytosphingosine, salts thereof, hydrates thereof, solvatesthereof, polymorphs thereof, optical isomers, optionally enantiomersand/or diastereomers and/or epimers, thereof, or mixtures of at leasttwo of the aforementioned sphingoid bases.

Optionally, the sphingoid base according to formula I is sphingosine,optionally D-sphingosine, D-erythro-sphingosine or(2S,3R,4E)-2-amino-4-octadecene-1,3-diol, according to the followingformula II-a:

Alternatively or in combination, the sphingoid base according to formulaI is dihydrosphingosine, optionally D-erythro-dihydrosphingosine or(2S,3R)-2-aminooctadecane-1,3-diol, according to the following formulaII-b:

Alternatively or in combination, the sphingoid base according to formulaI is phytosphingosine, optionally 4D-hydroxysphinganine,(2S,3S,4R)-2-aminooctadecane-1,3,4-triol, according to the followingformula II-c:

Alternatively or in combination, the sphingoid base according to formulaI is dehydrophytosphingosine, optionallyD-erythro-dehydrophytosphingosine or(8E)-2-aminooctadec-8-ene-1,3,4-triol or 4R-hydroxysphing-8E-enine,according to the following formula II-d:

Alternatively or in combination, the sphingoid base according to formulaI can be a non-natural isomer of sphingosine, dihydrosphingosine,phytosphingosine or dehydrophytosphingosine.

Optionally, the sphingoid base according to formula I can beD-erythro-C20-sphingosine according to the following formula II-e:

Alternatively or in combination, the sphingoid base according to formulaI can be D-threo-sphingosine according to the following formula II-f:

Alternatively or in combination, the sphingoid base according to formulaI can be L-threo-dihydrosphingosine according to the following formulaII-g:

Alternatively or in combination, the sphingoid base according to formulaI can be D-erythro-sphingosine according to the following formula II-h:

Alternatively or in combination, the sphingoid base according to formulaI can be D-threo-dihydrosphingosine according to the following formulaII-i:

Alternatively or in combination, the sphingoid base according to formulaI can be 3-deoxy-D-erythro-sphingosine according to the followingformula II-j:

Alternatively or in combination, the sphingoid base according to formulaI can be D-erythro-dihydrosphingosine according to the following formulaII-k:

Alternatively or in combination, the sphingoid base according to formulaI can be L-threo-sphingosine according to the following formula II-1:

Alternatively or in combination, the sphingoid base according to formulaI can be L-erythro-dihydrosphingosine according to the following formulaII-m:

Alternatively or in combination, the sphingoid base according to formulaI can be D-erythro-C16-sphingosine according to the following formulaII-n:

Alternatively or in combination, the sphingoid base according to formulaI can be 1-deoxy-D-erythro-dihydrosphingosine according to the followingformula III-a:

Alternatively or in combination, the sphingoid base according to formulaI can be 1-deoxymethylsphingosine according to the following formulaIII-b:

Alternatively or in combination, the sphingoid base according to formulaI can be 1-deoxy-D-erythro-sphingosine according to the followingformula III-c:

Alternatively or in combination, the sphingoid base according to formulaI can be monomethyl-D-erythro-sphingosine according to the followingformula III-d:

Alternatively or in combination, the sphingoid base according to formulaI can be galactosylsphingosine according to the following formula III-e:

Alternatively or in combination, the sphingoid base according to formulaI can be D-erythro-C20-sphingosine, L-erythro-sphingosine,D-threo-dihydrosphingosine (D-threo-sphinganine),L-threo-dihydrosphingosine (L-threo-sphinganine),1-deoxy-D-erythro-dihydrosphingosine, 1-deoxymethylsphingosine,monomethyl-D-erythro-sphingosine, glucosylsphingosine,D-erythro-dihydrosphingosine (D-erythro-sphinganine),L-erythro-dihydrosphingosine (L-erythro-sphinganine),D-threo-dihydrosphingosine (D-threo-sphinganine),L-threo-dihydrosphingosine (L-threo-sphinganine) or mixtures of at leasttwo of the aforementioned sphingoid bases.

In some aspects, a composition includes a sphingoid compound, butexcludes other compounds or compositions that function as an antiviralor otherwise reduce infection or inhibit one or more aspects of the lifecycle of a virus, optionally rhinovirus. Optionally, a compositionconsists of a sphingoid compound, optionally a sphingoid base,optionally sphingosine, and non-functional additives wherenon-functional means additives that alone do not reduce, treat, orprevent infection by a virus, optionally rhinovirus.

The inventors further identified on the basis of in vitro experimentsthat, surprisingly, the addition of an enzyme that catalyzes thegeneration or production of a sphingoid base, optionally sphingosine, islikewise suitable for treating or preventing a viral infection and isthus suited to preventing a viral infectious disease and/or to treatinga viral infection and/or viral infectious disease.

In some aspects of this disclosure, the active ingredient is an enzymethat catalyzes the generation or production of a sphingoid base,optionally of sphingosine. Optionally, the active ingredient isceramidase, illustratively but not limited to acid, neutral or alkalineceramidase. Alternatively or in combination, the active ingredient issphingosine-1-phosphate phosphatase. Furthermore, the active ingredientcan be a mixture of at least two of the enzymes mentioned in thisparagraph.

In some aspects, the active ingredient is an activator of an enzyme thatdirectly or indirectly catalyzes the generation or production of asphingoid base, optionally of sphingosine. The expression “activator” isto be understood in this connection in the context of the presentdisclosure to mean a compound capable of activating or stimulating suchan enzyme, optionally the catalytic activity thereof, in some way. Inparticular, the activator can be a compound that activates or stimulatesthe expression, optionally the transcription and/or the translation,and/or a post-translational modification, of the enzyme.

Optionally, the activator is a ceramidase activator and/orsphingomyelinase activator and/or a sphingosine-1-phosphate phosphataseactivator, or any combination thereof. For example, the activator can bean activator of acid ceramidase, of neutral ceramidase, of alkalineceramidase, a sphingomyelinase, or of sphingosine-1-phosphatephosphatase. Furthermore, the activator can be a mixture of at least twoof the activators mentioned in this paragraph.

In some aspects of this disclosure, the active ingredient is aninhibitor of an enzyme that directly or indirectly catalyzes thedegradation of a sphingoid base, optionally of sphingosine. Theexpression “inhibitor” is to be understood in this connection in thecontext of the present disclosure to mean a compound that inhibits orreduces the activity of the enzyme, optionally the catalytic activitythereof, in some way. Optionally, the inhibitor can be a compound thatinhibits or reduces the expression, optionally the transcription and/orthe translation and/or a post-translational modification, of the enzyme.

Optionally, the inhibitor is a sphingosine kinase inhibitor or aceramide synthase (CerS) inhibitor. For example, the inhibitor can be aninhibitor of sphingosine kinase 1, of sphingosine kinase 2, of ceramidesynthase 1, of ceramide synthase 2, of ceramide synthase 3, of ceramidesynthase 4, or ceramide synthase 5, of ceramide synthase 6, or anycombination thereof. Furthermore, the inhibitor can be a mixture of atleast two of the inhibitors mentioned in this paragraph.

The inhibitor, optionally ceramide synthase inhibitor, can be Fingolimodor a derivative thereof, optionally as disclosed in Schiffmann, et al.,Biochimie, 2012; 94(2):558-65, P053 as found in Turner, et al., NatureCommunication, 2018; Article number: 3165.

Optionally, the inhibitor is an inhibitor of sphingosine kinase 1 and/oran inhibitor of sphingosine kinase 2.

In some aspects, the inhibitor is a sphingosine kinase 1 inhibitor,optionally selected from the group consisting of(2R,3S,4E)-N-methyl-5-(4-pentylphenyl)-2-aminopent-4-ene-1,3-diol,(R)-(1-(4-((3-methyl-5-(phenylsulfonylmethyl)phenoxy)methyl)benzyl)pyrrolidin-2-yl)methanol,(2,2-dimethyl-4S-(1-oxo-2-hexadecyn-1-yl)-1,1-dimethylethylester-3-oxazolidinecarboxylic acid,(N′-[1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxyphenyl)-1H-pyrazole-5-carbohydrazide),Compound 82 (Amgen), amidine-based inhibitors such as VPC94075 andCB5468139, and mixtures of at least two of the aforementionedinhibitors.

Optionally, the inhibitor is a sphingosine kinase 2 inhibitor optionallyselected from the group consisting of ABC294640,[3-(2-aminoethyl)-5-[3-(4-butoxylphenyl)propylidene]thiazolidine-2,4-dione],synthetic sphingosine analogues SG12 and SG14, (R)-FTY720-OMe,(S)-2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide,(S)-2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)azetidine-1-carboximidamidehydrochloride, and mixtures of at least two of the aforementionedinhibitors.

In some aspects, the inhibitor is a sphingosine kinase 1 inhibitorand/or sphingosine kinase 2 inhibitor, optionally selected from thegroup consisting of SKI-II([2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole]), SK1-I((2R,3S,4E)-N-methyl-5-(4-pentylphenyl)-2-aminopent-4-ene-1,3-diol),MP-A08 and mixtures of at least two of the aforementioned inhibitors.

In principle, the sphingoid base and/or the active ingredient can beprepared for a local or systemic administration.

Optionally, the sphingoid base and/or the active ingredient is preparedfor an intravenous, intra-arterial, cutaneous, subcutaneous,percutaneous, intramuscular, inhalational, intravaginal, oral, nasal,pharyngeal, tracheal, pulmonary, or conjunctival administration. Inother words, the sphingoid base and/or the active ingredient may be usedfor an intravenous, intra-arterial, cutaneous, subcutaneous,percutaneous, intramuscular, inhalational, intravaginal, oral, nasal,conjunctival administration, or any combination thereof. Optionally, theadministration is by injection, spray, dry powder, inhalation or anycombination thereof.

Optionally, administration is an intravenous or nasal administration. Anintravenous administration may result in a systemic and thus extensiveprophylactic and/or therapeutic action. A nasal administration mayresult in a rapid onset of action and may directly target the site ofinfection of some viruses, illustratively rhinoviruses. Moreover, withboth forms of administration, a further advantage is that just a lowdose may be sufficient to achieve a desired action.

Optionally, the prophylaxis is, in the context of the presentdisclosure, a vaccination and/or an improvement in success ofvaccination against a viral infection and/or viral infectious disease.

The sphingoid base and/or the active ingredient can be prepared for anadministration in a dose of from 0.001 mg/kg body weight to 10 mg/kgbody weight, optionally 0.01 mg/kg body weight to 1 mg/kg body weight,optionally 0.1 mg/kg body weight, or be used for such an administrationat such dose.

Optionally, the viral infection and/or viral infectious disease is aviral infection and/or viral infectious disease in a human, i.e. a humanviral infection and/or human viral infectious disease.

Optionally, the viral infection and/or viral infectious disease iscoronavirus disease of 2019 (COVID-19), haemorrhagic fever, haemorrhagicfever with renal syndrome (HFRS), encephalitis, meningoencephalitis,tick-borne encephalitis (TBE), lymphocytic choriomeningitis, Lassafever, herpes simplex type 1, herpes simplex type 2, hepatitis B, herpeszoster, glandular fever, cytomegaly, acquired immunodeficiency syndrome(AIDS), severe acute respiratory syndrome, smallpox, rubella, hepatitisC, Zika fever, West Nile fever, dengue fever, yellow fever, Rift Valleyfever, sandfly fever, Marburg fever, Ebola, influenza, braininflammation, measles, mumps, respiratory virus disease, BK nephropathy,hepatitis C, poliomyelitis, viral meningitis, or myocarditis.

Optionally, the viral infection and/or viral infectious disease can be aviral infection and/or viral infectious disease which is/are broughtabout or caused by enveloped viruses, selected in particular from thegroup consisting of Arenaviridae, Herpesviridae, Retroviridae,Coronaviridae, Poxviridae, Togaviridae, Flaviviridae, Bunyaviridae,Filoviridae, Orthomyxoviridae, Paramyxoviridae and Rhabdoviridae.

The Coronaviridae can be selected from the group consisting ofColacovirus, Decacovirus, Duvinacovirus, Luchacovirus, Minacovirus,Minunacovirus, Myotacovirus, Nyctacovirus, Pedacovirus, Rhinacovirus,Setracovirus, Soracovirus, Sunacovirus, Tegacovirus, Embecovirus,Hibecovirus, Merbecovirus, Nobecovirus, Sarbecovirus, Brangacovirus,Brangacovirus, Igacovirus, Andecovirus, Buldecovirus, and Herdecovirus.Optionally, the Coronaviridae is a Betacoronavirus, optionally a Severeacute respiratory syndrome-related coronavirus, optionally a Severeacute respiratory syndrome coronavirus (SARS-CoV) or a Severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2), or other relatedmembers.

The Arenaviridae can be selected from the group consisting of Lassavirus, Junin virus and lymphocytic choriomeningitis virus (LCMV).

The Herpesviridae can be selected from the group consisting of herpessimplex virus, hepatitis B virus, varicella-zoster virus,cytomegalovirus and Epstein-Barr virus.

The Retroviridae can, for example, be the human immunodeficiency virusor the human T-lymphotropic virus.

The Poxviridae can, for example, be the smallpox virus.

The Togaviridae can, for example, be the rubella virus or the SemlikiForest virus.

The Flaviviridae can be selected from the group consisting of hepatitisC virus, Zika virus, West Nile virus, dengue virus, yellow fever virusand TBE virus.

The Bunyaviridae can be selected from the group consisting of RiftValley fever virus, sandfly fever virus and Hantaan virus.

The Filoviridae can, for example, be the Marburg virus or Ebola virus.

The Orthomyxoviridae can, for example, be selected from the groupconsisting of influenza virus A, influenza virus B and influenza virusC.

The Paramyxoviridae can be selected from the group consisting of Nipahvirus, human parainfluenza virus, measles virus and mumps virus.

The Rhabdoviridae can, for example, be the vesicular stomatitis virus orrabies virus.

In particular, the viral infection and/or viral infectious disease canbe a viral infection and/or viral infectious disease in a human, i.e. ahuman viral infection and/or human viral infectious disease, whichis/are brought about or caused by non-enveloped viruses, optionallyAdenoviridae, Polyomaviridae, Papillomaviridae, Hepeviridae, orrhinovirus.

The Adenoviridae can, for example, be the human adenovirus.

The Polyomaviridae can, for example, be the BK virus.

The Papillomaviridae can, for example, be the human papillomavirus.

The Hepeviridae can, for example, be the hepatitis E virus.

The Picornaviridae can, for example, be the poliovirus, coxsackievirusor the enterovirus.

The virus can, for example, be human rhinovirus.

The virus can, for example, be a SARS-CoV-2.

In some aspects, the viral infection and/or viral infectious disease isa viral infection and/or viral infectious disease in a non-human animal,such as, for example, a dog, a cat, a horse, a cow, a pig, a bird, afish, a reptile, an amphibian, a mollusc, an insect or a spider.

The viral infection and/or viral infectious disease is/are optionallyCOVID-19, monkeypox, African horse sickness, African swine fever, equineinfectious anaemia (swamp fever), infectious salmon anaemia, Aujeszky'sdisease in domestic cattle and/or domestic pigs, bluetongue disease,bovine herpesvirus type 1 infections, bovine viral diarrhoea, commoncold, Ebola virus infection, epizootic haemorrhage in deer, epizootichaematopoietic necrosis, enzootic leukosis in cattle, avian influenza(bird flu), West Nile virus infection in birds and/or horses, koiherpesvirus infection in carp, foot-and-mouth disease, Newcastledisease, peste des petits ruminants (ovine rinderpest), equineencephalomyelitis, sheep and goat pox, Rift Valley fever, rinderpest,swine fever (European swine fever), vesicular stomatitis, Taurasyndrome, rabies, swine vesicular disease, viral haemorrhagicsepticaemia in Salmonidae, white spot disease in crustaceans, oryellowhead disease, optionally in shrimp and prawns.

Optionally, the viral infection and/or viral infectious disease can be aviral infection and/or viral infectious disease that is/are broughtabout or caused by the monkeypox virus, African horse sickness virus(AHS), African swine fever virus, equine infectious anaemia virus,infectious salmon anaemia virus, Aujeszky's disease virus, bluetonguevirus, bovine herpesvirus type 1, bovine viral diarrhoea virus, Ebolavirus, epizootic haemorrhagic disease virus (EHDV), rhabdovirus, bovineleukaemia virus, influenza virus, West Nile virus, koi herpesvirus,foot-and-mouth disease virus, Newcastle disease virus, Capripoxvirusovis, Capripoxvirus caprae, phleboviruses, rinderpest virus, vesicularstomatitis virus, Taura syndrome virus, rabies virus, swine vesiculardisease virus, white spot disease virus or yellowhead virus.

In some aspects, the viral infection and/or viral infectious disease isa viral infection and/or viral infectious disease of a plant, i.e. aplant viral infection and/or plant viral infectious disease, selected inparticular from the group consisting of tomato spotted wilt disease,impatiens necrotic spot disease, pepino mosaic disease, zucchini yellowmosaic disease, papaya ringspot disease, watermelon mosaic disease,Moroccan watermelon mosaic disease, potato virus A disease, tobaccorattle disease, potato virus Y disease, squash mosaic disease, cucumbergreen mottle mosaic disease, tobacco ringspot disease, tomato ringspotdisease, tobacco mosaic disease and tobacco necrosis.

Optionally, the viral infection and/or viral infectious disease can be aviral infection and/or viral infectious disease in a plant that is/arebrought about or caused by the tomato spotted wilt virus, impatiensnecrotic spot virus, pepino mosaic virus, zucchini yellow mosaic virus,papaya ringspot virus, watermelon mosaic virus, Moroccan watermelonmosaic virus, potato virus Y, potato virus A, tobacco rattle virus,squash mosaic virus, cucumber green mottle mosaic virus, tobaccoringspot virus, tomato ringspot virus, tobacco mosaic virus or tobacconecrosis virus.

In some aspects, provided is a medicament (drug) or a pharmaceuticalcomposition for application or use in the prophylaxis and/or therapy ofa viral infection and/or viral infectious disease or for application oruse in disinfection.

The medicament or the pharmaceutical composition comprises a sphingoidcompound and/or an active ingredient that influences, in particularactivates or inhibits, the degradation or the generation of a sphingoidbase, optionally of sphingosine, in vivo.

Optionally, the medicament or the pharmaceutical composition furthercomprises pharmaceutically acceptable carriers for the sphingoidcompound and/or the active ingredient. The carrier can, for example, beselected from the group consisting of water, acetone, ethanol, ethyleneglycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, liposomes, mineral oil and mixtures of at least twoof the carriers mentioned.

Furthermore, the medicament or the pharmaceutical composition canfurther include a surface-active substance, such as, for example,n-octyl-β-D-glucopyranoside (OGP).

In principle, the medicament or the pharmaceutical composition can beformulated for a local or systemic administration or be present in alocal or systemic form of administration.

In some aspects, the medicament or the pharmaceutical composition isformulated as a solution, a colloidal dispersion, an emulsion(oil-in-water emulsion or water-in-oil emulsion), a suspension, a cream,a lotion, a gel, a foam, a spray, an aerosol, an ointment, tablets,drops or a suppository. In other words, the medicament or thepharmaceutical composition is optionally present in the form of asolution, a colloidal dispersion, an emulsion (oil-in-water emulsion orwater-in-oil emulsion), a suspension, a cream such as skin cream, alotion, a gel, a foam, a spray such as nasal spray, an aerosol, anointment, tablets, drops or a suppository.

Optionally, the medicament or the pharmaceutical composition is a skincream, i.e. a cream for application to the skin or mucosa, or amedicament/pharmaceutical composition prepared for a nasaladministration, or a medicament/pharmaceutical composition prepared forocular administration. Optionally, the medicament or the pharmaceuticalcomposition can be a nasal spray, a nasal ointment or nasal drops.Optionally, the medicament or pharmaceutical composition can be an eyedrop or other known method.

In some aspects, the medicament or the pharmaceutical composition caninclude a proportion of the sphingoid base and/or the active ingredientof from 0.01% by weight to 100% by weight, optionally 0.1% by weight to30% by weight, and optionally 1% by weight to 10% by weight, based onthe total weight of the medicament or the pharmaceutical composition.

With regard to further features and advantages of the sphingoid baseand/or the active ingredient, full reference is made to the remarks madein the context of the disclosure above in order to avoid repetition. Thefeatures and advantages described there with regard to the sphingoidbase and/or the active ingredient also apply mutatis mutandis to amedicament or drug or a pharmaceutical composition.

Also provided are the use of a sphingoid base as a food or foodsupplement.

The food or food supplement includes a sphingoid base and/or an activeingredient.

In the context of the present disclosure, the term “food” is to beunderstood to mean a food which serves for the nutrition of a human ornon-human animal.

In the context of the present disclosure, the expression “foodsupplement” is to be understood to mean a product for the supplementalnutrition of a human and/or for the increased or improved supply ofnutrients or active ingredients to human metabolism.

With regard to further features and advantages of the sphingoid baseand/or the active ingredient, full reference is made to the remarks madein the context of the disclosure above in order to avoid repetition. Thefeatures and advantages described elsewhere herein with regard to thesphingoid base and/or the active ingredient also apply mutatis mutandisto a food or food supplement as also provided herein.

Also provided herein is a feed or feed supplement. The feed or feedsupplement includes a sphingoid base and/or an active ingredient asprovided herein. In the context of this disclosure, the term “feed” isto be understood to mean a product for the nutrition of non-humananimals, optionally non-human mammals, birds or fishes, i.e. a so-calledanimal food. For example, the feed can be a feed for fishes, optionallyaquarium fishes. Alternatively, the feed can be a feed for dogs, cats,horses, cows, pigs, or other non-human animal.

As used herein, the term “feed supplement” is to be understood to mean aproduct for the supplemental nutrition of non-human animals, optionallynon-human mammals, birds or fishes, and/or for the increased or improvedsupply of nutrients or active ingredients to the metabolism of non-humananimals, optionally non-human mammals, birds or fishes. For example, thefeed supplement can be a feed supplement for fishes, optionally aquariumfishes, or other non-human animals.

Also provided herein is a plant protection agent. The plant protectionagent includes a sphingoid base and/or an active ingredient as otherwisedescribed herein. For example, the plant protection agent can be a plantprotection agent for cultivated plants, illustratively wheat, rapeseed,tomatoes, or the like. With regard to further features and advantages ofthe sphingoid base and/or the active ingredient, full reference is madeto the remarks made in the context of the disclosure above in order toavoid repetition. The features and advantages described there withregard to the sphingoid base and/or the active ingredient also applymutatis mutandis to a plant protection agent as provided herein.

Optionally, as provided herein a sphingoid base of formula I is used forapplication or use in the inhibition or hindering of bacteriophages orfor application or use in the stabilization and/or spreading of abacterial flora, optionally healthy bacterial flora, or for applicationor use in the avoidance of the formation of resistant bacterial strains.

Optionally, medicament or the pharmaceutical composition is an activeingredient that influences, optionally activates or inhibits, thedegradation or the generation of a sphingoid base, optionally ofsphingosine, in vivo for application or use in the inhibition orhindering of bacteriophages or for application or use in thestabilization and/or spreading of a bacterial flora, optionally healthybacterial flora, or for application or use in the avoidance of theformation of resistant bacterial strains.

The above-mentioned bacterial flora can, for example, be a skin ormucosa, optionally intestinal mucosa. Furthermore, the skin can be ahuman skin or a skin of a non-human animal, optionally a non-humanmammal or a bird.

With regard to further features and advantages of the sphingoid baseand/or the active ingredient, full reference is made to the remarks madein the context of the disclosure above in order to avoid repetition. Thefeatures and advantages described there with regard to the sphingoidbase and/or the active ingredient also apply mutatis mutandis to theapplication or use of the sphingoid base and/or the active ingredientfor application or use in the inhibition or hindering of bacteriophagesor for application or use in the stabilization and/or spreading of abacterial flora, optionally healthy bacterial flora, or for applicationor use in the avoidance of the formation of resistant bacterial strains.

According to some aspects provided optionally for in vitro use is asphingoid base of the following formula I for the inhibition orhindering of bacteriophages or for the stabilization and/or spreading ofa bacterial flora, optionally healthy bacterial flora, or for theavoidance of the formation of resistant bacterial strains.

The above-mentioned bacterial flora can, for example, be a skin ormucosa, optionally intestinal mucosa. Furthermore, the skin can be ahuman skin or a skin of a non-human animal, optionally a non-humanmammal or a bird.

With regard to further features and advantages of the sphingoid baseand/or the active ingredient, full reference is made to the remarks madein the context of the disclosure above in order to avoid repetition. Thefeatures and advantages described herein with regard to the sphingoidbase and/or the active ingredient also apply mutatis mutandis to the useof a sphingoid base and/or an active ingredient for in vitro use.

Further features and advantages of the disclosure are revealed by thefollowing description of preferred embodiments in the form of exemplaryembodiments, the associated figures and the claims. The embodimentsdescribed below merely serve for further elucidation and for betterunderstanding of the disclosure and are in no way to be understood aslimiting.

EXPERIMENTAL SECTION

1. Methods and Materials

1.1 Mice and Tamoxifen Treatment

For in vivo analyses, Asah1^(−/−) mice, from which acid ceramidase wasgenetically removed, were used. Asah1^(−/−) mice were on a C57BL/6Jbackground. As control animals, sibling animals with the genotypeAsah1^(+/+) were used. Inducible ceramidase-deficient animals were used(Cre⁺ Asah1^(fl/fl)). Inducible ceramidase-deficient animals expressed aCre recombinase under the tamoxifen promoter and the gene Asah1^(−/−)between two LoxP sites. Administration of tamoxifen thus led to theexpression of the recombinase, which then removed the Asah1 gene.Tamoxifen was dissolved in corn oil. Eight, six and four days before anexperiment, Cre⁻×Asah1^(fl/fl)) control animals and Cre⁺ Asah1^(fl/fl)animals were treated intraperitoneally with, in each case, 4 mg oftamoxifen (in 100 μl).

1.2 Cell Lines, Bone Marrow Macrophages and Human T Cells

Raw264.7 and Hela cells were purchased from ATCC. Vero cells and MC57cells came from the Ontario Cancer Institute. Macrophages were generatedby isolating bone marrow from mice and culturing said bone marrow withM-CSF for 9 days. Cells were then plated out onto a new cell cultureplate and the experiment was started on the next day. Human T cells weregenerated by using EDTA-blood samples from healthy donors. CD8 T cellswere eliminated, and the remaining CD4 T cells were activated and werecultured until the start of the experiment.

1.3 Viruses

The LCMV strain WE and the VSV strain Indiana were obtained from thelaboratory of Prof Zinkernagel (Institute of Experimental Immunology,Zurich, Switzerland). HSV-1 came from Prof Beate Sodeik (Institute ofVirology, Hanover, Germany). Influenza A came from Prof. MatthiasTenbusch (Institute of Virology, Erlangen, Germany). The Friendretrovirus came from Prof Ulf Dittmer (Institute of Virology, Essen,Germany). The HIV came from Prof. Hendrik Streeck (Institute for HIVResearch). Rhinovirus was from ATCC.

1.4 Reagents

D-erythro-Sphingosine (C18) from Avanti Polar Lipids (860490-25 mg) wasused as a sphingoid base. Ceramidase (E 9030-100MUN) andsphingomyelinase (58633-25UN) were purchased from Sigma. Sphingosinekinase inhibitors, MP A08 (CAS #219832-49-2; cat. No. 5803) and SKII(CAS #: 312636-16-1; cat. No. 2097), came from Tocris.

1.5 Infection of Cells and Animals

For in vitro infection, the virus was added to the cell cultures. Afterincubation for one hour, the supernatant was washed away and freshmedium was added. The supernatant was removed at different times afterinfection. For in vitro infections with HSV-1, cell cultures werepre-treated on ice at 4° C. for 20 minutes, and virus was added underthese conditions and incubated for one hour. The supernatant wasremoved, new medium was added, and the cell cultures were stored at 37°C. until analysis. This approach is known as synchronized infection. Foran intravenous infection, the virus was injected in 100 μl into the tailvein. For an intranasal infection, the virus was pipetted, in 10 μleach, into each nares of the nose.

1.6 Determination of Virus Propagation by Means of Immunofluorescence

To be able to detect viruses in cells and organs, immunofluorescence wasused. For in vitro experiments, cells were seeded into 24-well platescontaining coverslips. After 24 hours, the cells were appropriatelyinfected and were fixed after incubation and labelled with antibodies.Organs from infected animals were flash-frozen. 8 μm thick sections wereaccommodated on a slide. The sections were fixed and viruses were thenvisualized by means of virus-specific antibodies. The following primaryantibodies were used: anti-LCMV-NP antibody (clone VL4);anti-HSV-1-capsid (SY4563, anti-capsid). Alternatively, virus directlylabelled with a dye (e.g. CFSE) was used.

1.7 Determination of VSV Virus Propagation

To measure the influence of sphingosine on the propagation of thevesicular stomatitis virus (VSV), Vero cells were seeded in a 24-wellplate. Once the cells had grown to confluence, ceramidase orsphingomyelinase was added. Thereafter, the cell lawn was infected withthe vesicular stomatitis virus (VSV) (100 PFU/well). After one hour,methylcellulose was added so that virus was able to spread only bycell-cell interaction. The result of this was that only cells inimmediate proximity were infected and thus an initial infection wasvisible via holes in the cell lawn (=plaques). After 24 hours, thenumber of plaques was ascertained.

1.8 Determination of LCMV in Organs and Cell Supernatant by Means ofPlaque Assay

To determine virus amount, cell culture supernatant or homogenized organlysate was titrated in a 24-well plate and soluble MC57 cells (150 000cells/well) were then added. After 4 hours, MC57 cells were able toadhere, and methylcellulose was added. After a further 48 hours, thecell lawn was analyzed for LCMV plaques using an anti-LCMV-NP antibody(clone VL4). The plaques were counted and thus the number of infectiousparticles per ml in the supernatant or lysate was determined.

1.9 Determination of IAV in Lungs

To determine the virus amount of the influenza A virus (IAV) fromhomogenized organ lysate, MDCK2 cells were seeded in a 24-well plate. Onthe next day, when the cells had grown to confluence, the organ lysateswere diluted and added. After two hours, methylcellulose was added sothat virus was able to spread only by cell-cell interaction. The resultof this was that only cells in immediate proximity were infected andthus an initial infection was visible via holes in the cell lawn(=plaques). After 24 hours, the number of plaques was ascertained andthus the number of infectious particles per ml in the lysate wasdetermined.

1.10 Determination of Friend Virus-Infected Cells

For the analysis of the Friend virus-infected cells, spleens and bloodwere analyzed. The spleens were mechanically crushed and the cells ofthe spleens were isolated after filtration. Cells and blood were stainedwith anti-B220 (label for B cells) and anti-Terr119 (label for erythroidprogenitor cells) and subsequently fixed with 2% formalin and then lysedwith saponin. Infected cells were ascertained by the expression ofwasabi.

1.11 Survival Experiments

To analyze the effect of sphingosine on the course of infection,survival experiments were carried out. To this end, animals wereinfected with a dose sublethal for WT animals. Animals were checkeddaily and were killed at relevant termination criteria and counted asdead. The termination criteria were: body weight, general health,spontaneous behavior and clinical findings.

1.12 P24 ELISA and HIV Infection

PBMCs (peripheral blood mononuclear cells) were purified from EDTA-bloodsamples from healthy donors (Streeck et al., 2007). CD8 T cells wereeliminated using CD8 MicroBeads (Miltenyi Biotec) and the remainingcells were activated using 1 μg/ml phytohaemagglutinin (PHA). Activatedcells were transferred into a 96-well plate and treated with thesubstances to be tested. Thereafter, the cells were infected with 63ng/ml HIV-1 JR-CSF virus for one hour. Using the HIV-1 Gag p24Quantikine ELISA Kit (R&D Systems), the infection rate was measured ondays 4 and 7 after infection. For the evaluation, the NanoQuant InfiniteM200 (Tecan) was used.

1.13 Production of Sphingosine-Containing Liposomes

A thin film of poly(vinyl alcohol) PVA was applied to glass iBidiplates. The fat mix was distributed on the plates and the liposomes wereincubated at 60° C. for one hour. The fat concentration in the liposomeswas as follows:

Control Ch:PC:Sm (33:33:33 molar) + liposomes 0.05 mol % BODIPY_PCCeramide Ch:PC:Sm:Cer (33:33:24:10 mol/%) + liposomes 0.05 mol %BODIPY_PC Sphingosine Ch:PC:Sm:Sph (33:33:24:10 mol/%) + liposomes 10.05 mol % BODIPY_PC Sphingosine Ch:PC:Sm:Sph (33:33:14:20 mol/%) +liposomes 2 0.05 mol % BODIPY_PC1.14 Statistical Analysis

The means were compared using an unpaired two-tailed Student's t-test.The data were presented as mean±SEM. The statistical significance levelwas defined at p<0.05. Significant differences were marked in the graphwith “*”.

2. Investigations

2.1

FIG. 1 is a schematic depiction of the natural metabolism ofsphingomyelin. Sphingomyelin (SM) is converted into ceramide (CER) bysphingomyelinase (SMase). Ceramide is converted into sphingosine (SPH)by ceramidase (CERase). Sphingosine is phosphorylated by sphingosinekinase (SPH kinase) to form sphingosine-1-phosphate (SPH-1-P). Alsoillustrated are exemplary enzymes that can alter the concentration ofsphingosine as a result of activation or inhibition wheresphingomyelinase is denoted (SMase), ceramidase is denoted (CERase), andsphingosine kinase is denoted (SPH-kinase).

2.2

Raw264.7 cells were treated with 250 μM D-erythro-sphingosine for 10minutes and then infected with the lymphocytic choriomeningitis virus(LCMV) (MOI. 0.01). After incubation for one hour, the medium waschanged and virus production was measured by means of plaque assay after6, 18 and 48 hours. In this case, it was possible to demonstrate thatsphingosine inhibits the propagation of LCMV. The results obtained aredepicted graphically in FIG. 2. Shown is the mean±SEM, n=3, *p<0.05student's T test.

2.3

Raw264.7 cells were treated with ceramidase (250 mUnits/ml) for 10minutes and then infected with the lymphocytic choriomeningitis virus(LCMV) (MOI. 0.01). After incubation for one hour, the medium waschanged and virus production was measured by means of plaque assay after6, 18 and 48 hours. In this case, it was possible to demonstrate thatceramidase inhibits the propagation of LCMV. The results obtained aredepicted graphically in FIG. 3. Shown is the mean±SEM, n=5, *p<0.05student's T test.

2.4

Raw264.7 cells were treated with sphingomyelinase (6.5 U/ml) for 10minutes and then infected with the lymphocytic choriomeningitis virus(LCMV) (MOI 0.01). After incubation for one hour, the medium was changedand virus production was measured by means of plaque assay after 6, 18and 24 hours. In this case, it was possible to demonstrate thatsphingomyelinase inhibits the propagation of LCMV. The results obtainedare depicted graphically in FIG. 4. Shown is the mean±SEM, n=3, *p<0.05student's T test.

2.5

Hela cells were treated with the sphingosine kinase inhibitor SKII (10μM and 100 μM) for 10 minutes. Cells were infected with the lymphocyticchoriomeningitis virus (LCMV) (MOI 1). After 24 hours, virus-infectedcells were measured by means of immunofluorescence. In this case, it waspossible to demonstrate that sphingosine kinase inhibition inhibits thespreading of the virus. The results obtained are depicted asimmunofluorescence images in FIG. 5 where white are the LCMV-infectedcells in cultures without sphingosine kinase inhibitor (0 μM), with 10μM sphingosine kinase inhibitor and with 100 μM sphingosine kinaseinhibitor (n=5).

2.6

Macrophages were obtained from the bone marrow of WT mice andceramidase-deficient mice (Asah1^(−/−)) and then infected with herpessimplex virus (HSV-1) (MOI 5). After 4 hours, the propagation of thevirus was measured by means of immunofluorescence. In this case, it waspossible to demonstrate that ceramidase deficiency promotes thespreading of the virus. The results obtained are depicted asimmunofluorescence images in FIG. 6. Shown in white are HSV-1 in nucleiof infected wild-type (WT) cells and ceramidase-deficient (Asah1^(−/−))cells (n=4).

2.7

WT mice and ceramidase-deficient mice (Asah1^(−/−)) were infectedintravenously with herpes simplex virus (HSV-1) (7×10⁷ PFU/mouse). After4 hours, the propagation of the virus was measured in the liver by meansof immunofluorescence. In this case, it was possible to demonstrate thatceramidase deficiency promotes the spreading of the virus in the liverof infected animals. The results obtained are depicted asimmunofluorescence images in FIG. 7. Shown in white are HSV-1 in livercells of infected wild-type (WT) animals and ceramidase-deficient(Asah1^(−/−)) animals (n=6).

2.8

Control mice and inducible ceramidase-deficient mice were treated withtamoxifen in order to switch off the ceramidase. The animals were leftuntreated or infected intravaginally with herpes simplex virus (HSV-1)(2×10⁷ PFU/mouse). The survival of the animals was ascertained. In thiscase, it was possible to demonstrate that ceramidase deficiency reducesthe survival of the infected animals. The results obtained are depictedgraphically in FIG. 8. p=0.0004; infected control animals (Cre⁻Asah1^(fl/fl)) n=12; infected ceramidase-deficient animals (Cre⁺Asah1^(fl/fl)) n=11; uninfected control animals (Cre⁻ Asah1^(fl/fl))n=5; uninfected ceramidase-deficient animals (Cre⁺ Asah1^(fl/fl)) n=8.

2.9

WT mice and ceramidase-deficient mice (Asah1^(−/−)) were infectedintravenously with the lymphocytic choriomeningitis virus (LCMV) (2×10⁶PFU/mouse). After 48 hours, the propagation of the virus was measured inthe liver by means of immunofluorescence. In this case, it was possibleto demonstrate that ceramidase deficiency promotes the spreading of thevirus in the liver of infected animals. The results obtained aredepicted as immunofluorescence images in FIG. 9. Shown in white are LCMVin liver cells from infected wild-type (WT) and ceramidase-deficient(Asah1^(−/−)) animals (n=6).

2.10

WT mice, ceramidase-deficient mice (Asah1^(−/−)) and interferon-alphareceptor-deficient mice (Ifnar^(−/−)) were infected intravenously withthe lymphocytic choriomeningitis virus (LCMV) (2×10⁶ PFU/mouse). After48 hours, the number of infectious virus particles was measured indifferent organs. In this case, it was possible to demonstrate thatceramidase inhibits the propagation of LCMV in all the organs tested.The inhibitory action of the ceramidase was just as strong as that ofthe strongest antiviral gene currently known, the type I interferonreceptor (Ifnar). The results obtained are depicted graphically in FIG.10. Shown is the mean±SEM, WT n=6; Asah1^(−/−)=4; Ifnar^(−/−)=4,*p<0.05; **p<0.01; #p<0.001; ##p<0.0001; student's T test.

2.11

Control mice and inducible ceramidase-deficient mice were treated withtamoxifen in order to switch off the ceramidase. The animals were leftuntreated or infected intravenously with the lymphocyticchoriomeningitis virus (LCMV) (2×10⁴ PFU/mouse). In this case, it waspossible to demonstrate that ceramidase deficiency reduces the survivalof the infected animals. The results obtained are depicted graphicallyin FIG. 11. p<0.0001; infected control animals (Cre⁻ Asah1^(fl/fl)) n=8;infected ceramidase-deficient animals (Cre⁺ Asah1^(fl/fl)) n=7;uninfected ceramidase-deficient animals (Cre⁺ Asah1^(fl/fl)) n=4.

2.12

Confluently grown Vero cells were treated with 0, 10 and 50 mU/200 μlceramidase for 10 minutes and then infected with the vesicularstomatitis virus (VSV) (100 PFU/well in a 24-well plate). After onehour, methylcellulose was added. After 24 hours, the number of plaques(due to holes in the cell lawn that are caused by the virus) wasascertained. In this case, it was possible to demonstrate that theceramidase inhibits the propagation of VSV. The results obtained aredepicted graphically in FIG. 12. Shown is the mean±SEM, 0 n=10, 10 n=8,50 n=4, **p<0.01; ##p<0.0001; student's T test.

2.13

Confluently grown Vero cells were treated with 0, 250 and 1250 mU/200 μlsphingomyelinase for 10 minutes and then infected with the vesicularstomatitis virus (VSV) (100 PFU/well in a 24-well plate). After onehour, methylcellulose was added. After 24 hours, the number of plaques(due to holes in the cell lawn that are caused by the virus) wasascertained. In this case, it was possible to demonstrate that thesphingomyelinase inhibits the propagation of VSV. The results obtainedare depicted graphically in FIG. 13. Shown is the mean±SEM, 0 n=8, 250n=8, 1250 n=4, #p<0.001, student's T test.

2.14

Control mice and inducible ceramidase-deficient mice were treated withtamoxifen in order to switch off the ceramidase. The animals wereinfected intravenously with the vesicular stomatitis virus (VSV) (2×10⁶PFU/mouse). In this case, it was possible to demonstrate that ceramidasedeficiency reduces the survival of the infected animals. The resultsobtained are depicted graphically in FIG. 14. p=0.0041; infected controlanimals (Cre-Asah1^(fl/fl)) n=7; infected ceramidase-deficient animals(Cre⁺ Asah1^(fl/fl)) n=8.

2.15

WT mice and ceramidase-deficient mice (Asah1^(−/−)) were infectedintranasally with the influenza A virus (IAV) (2.5×10⁵ PFU/mouse). After3 days, the number of infectious virus particles was measured in thelung. In this case, it was possible to demonstrate that ceramidaseprevents the propagation of IAV. The results obtained are depictedgraphically in FIG. 15. Shown is the mean±SD, WT n=8, Asah1^(−/−) n=6,*p<0.05, student's T test.

2.16

WT mice and ceramidase-deficient mice (Asah1^(−/−)) were infectedintravenously with the Friend virus (FV) (2×10⁴ SFFU/mouse). After 6days, the percentage of infected erythroid progenitor cells wasmeasured. In this case, it was possible to demonstrate that ceramidaseprevents the propagation of FV. The results obtained are depictedgraphically in FIG. 16. Shown is the mean±SD, WT n=10, Asah1^(−/−) n=6,*p<0.05, student's T test.

2.17

Human T cells were isolated from blood and infected with 63 ng/ml HIV-1JR-CSF. One group was additionally treated with 50 μM of the sphingosinekinase inhibitor MP A08. After 7 days, the amount of HIV-P24 in thesupernatant of the cultures was quantified by means of ELISA. In thiscase, it was possible to demonstrate that sphingosine kinase inhibitionprevents the propagation of HIV. The results obtained are depictedgraphically in FIG. 17. Shown is the mean±SEM, n=9, *p<0.05, student's Ttest.

2.18

Liposomes were enriched by means of ceramide or sphingosine. Controlliposomes, ceramide liposomes and sphingosine liposomes were incubatedwith fluorescent lymphocytic choriomeningitis virus (LCMV). After 10minutes, the binding of the virus was measured using a flow cytometer.In this case, it was possible to demonstrate that sphingosine-containingliposomes can bind a virus rapidly. The results obtained are depictedgraphically in FIG. 18. Shown is the mean±SD, n=3, ns—not significant,#p<0.001, student's T test.

2.19

Liposomes were enriched by means of ceramide or sphingosine. Controlliposomes, ceramide liposomes and sphingosine liposomes were incubatedwith the fluorescent lymphocytic choriomeningitis virus (LCMV). After 10minutes, the binding of the virus was measured by means of confocalmicroscopy. In this case, it was possible to demonstrate thatsphingosine-containing liposomes can bind a virus rapidly. The resultsobtained are depicted as micrographs in FIG. 19 (n=3).

3. The Effect of Sphingosine on Rhinoviral (RV) Infections

3.1

Sphingosine (from Avanti Polar Lipids, CAS #: 123-78-4, cat. No. 860490)was added to human epithelial Hela cells at different concentrations anddifferent times prior or after viral infections. Hela cells werecultured in 24 well plates in DMEM supplemented with 2 mM L-glutamine, 1mM sodium pyruvate, 100 μM nonessential amino acids, 100 U/mLpenicillin, 100 μg/mL streptomycin (all from Invitrogen) and 10% fetalcalf serum (PAA Laboratories GmbH, Coelbe, Germany) and grown until theyreached approximately 70% density. Cells were washed twice in DMEMsupplemented with 2% FCS and recultured in DMEM/2% FCS. 5×10⁵ Hela cellswere infected with 10⁵ PFU of rhinovirus strains RV2 (minor strain) orRV14 (major strain). Rhinoviruses were obtained from ATCC. Sphingosinewas added at 10 μM or 20 μM final concentration 10 min prior theinfection (before infection, b.i.) with rhinoviruses or 60 min or 240min after infection (a.i.) with rhinoviruses. Sphingosine was dilutedfrom a 20 mM stock in 10% octylglucopyranoside (OGP) in distilled water.Thus, controls were 0.005% or 0.01% OGP final concentrations. Furthercontrols were infected, but not further treated. The samples wereincubated for 4 hrs at 33° C. after initiating the infection, thesupernatant was removed, DMEM/10^(%) FCS was added and the cells wereincubated for an additional 4 days. Cell death was determined as ameasurement for the infection by staining the cells with Trypan Blue.Rhinoviruses are cytotoxic and the number of dead cells accuratelyreflects the rhinoviral infection. We determined the percentage of deadcells by counting 500 cells/sample using a cell culture microscopy.

As illustrated in FIG. 20 sphingosine prevented the infection with RV2and RV14 at 10 μM and 20 μM. An increase of the sphingosineconcentration to 20 μM did not significantly increase the inhibition ofviral infections by sphingosine. Sphingosine prevented the infection ifadded prior to the virus or after infection with the virus up to 4 hrs.Sphingosine itself had no effect on the cells. OGP did not alter theinfection. Shown is the percentage of dead cells 4 days after initiatingthe infection±SD, n=5 each, ***p<0.001. ANOVA.

3.2

Sphingosine (SPH) was applied into the nose of wild-type mice and theconcentration of sphingosine on the epithelial cell surface wasdetermined after 1, 4 and 8 hrs (a.i.: after injection of sphingosine).To this end, 10 μL of a 10 μM or a 100 μM sphingosine solution wasapplied into the nostrils of wild-type mice. Controls received thesolvent, i.e. octylglucopyranoside at the same concentration used whensphingosine was applied, or were left untreated. Mice were slightly andvery briefly anesthetized with ether. For the intranasal application weused a blunt-end 30 g needle that was covered with a thin plastic film.The blunt-ended needle was inserted into the nostrils approximately 2mm. Mice were sacrificed after the indicated time, the nasal bone wasremoved, placed on a 30° C. pre-warmed plastic plate and an area of 2mm×2 mm was immediately incubated with 0.001 units of sphingosine kinase1 (#6068-SK-010, R&D) in 4 μl of 150 mM sodium acetate (pH 7.4), 1 μMATP, and 10 μCi [³²P]γATP/sample. Controls were incubated with the samebuffer without sphingosine kinase or were left untreated. Thesphingosine kinase reaction was terminated by adding 100 μl H₂O,followed by the addition of 20 μl 1N HCl, 800 μl CHCl₃:CH₃OH:1N HCl(100:200:1, v:v:v), and 240 μl each of CHCl₃ and 2 M KCl. The lowerphase was collected, dried, dissolved in 20 μL CHCl₃:CH₃OH (1:1, v/v),and separated on Silica G60 TLC plates with CHCl₃:CH₃OH:acetic acid:H₂O(90:90:15:5, v:v:v:v) as a developing solvent. The TLC plates wereanalyzed with a phosphoimager. Surface sphingosine levels weredetermined with a standard curve of C18-sphingosine.

The assays were performed without washing the mucosa on the nasal bone,i.e. immediately after removal, and, in addition, after extensivewashing the specimen in PBS to remove mucus from the epithelial cellsurface.

The results are depicted in FIG. 21 illustrating that application ofsphingosine into the nose of wild-type mice results in a dose-dependentaccumulation of sphingosine in the mucosa. Washing the mucosa prior tothe in situ kinase assay reduces sphingosine accumulation in the nasalspecimen on top of the epithelial cell layer indicating that mostsphingosine remains in the mucus on top of the epithelial cell layer.Shown are the surface concentration, mean SD, n=4, ***p<0.001, ANOVA.

3.3

Sphingosine was applied into the nose of wild-type mice as in 3.2. 10 μLof a 10 μM or a 100 μM sphingosine (SPH) solution was administered intothe nostrils of wild-type mice. Controls received the solvent, i.e.octylglucopyranoside at the same concentration used when sphingosine wasapplied, or were left untreated. Mice were anesthetized prior toapplication of sphingosine. Mice were sacrificed after 8 hours or 24hours, the nasal bone was removed and incubated in Trypsin solution for10 min to release and isolate epithelial cells. Cells were washed twicein H/S (20 mM HEPES, 132 mM NaCl, 5 mM KCl, 1 mM CaCl₂), 0.7 mM MgCl₂,0.8 mM MgSO₄, pH 7.4), stained with Trypan Blue (0.2% finalconcentration) and dead cells were counted in aliquots of 500 cells.Given is the percentage of dead cells±SD from 4 independent experimentseach; *p<0.05, ANOVA. The results show that sphingosine at the applieddoses had no toxic effects on epithelial cells of the nose.

3.4

Mice were infected with 10³ PFU rhinovirus strain 2 (RV2) in 10 μL PBS.The virus was applied directly into the nose using a blunt ended,plastic coated 30 g needle, that was inserted into the noseapproximately 2 mm. Sphingosine (SPH) was applied into the nose of thewild-type mice at a concentration of 100 μM solution in PBS in a volumeof 10 μL. Sphingosine was applied either 10 min prior to rhinovirusinfection (b.i.) or 1 hr, 4 hrs and 8 hrs after the infection (a.i.).Controls received the solvent, i.e. octylglucopyranoside (OGP) at thesame concentration than used when sphingosine was applied, or were leftuntreated, but infected. Solutions were applied as in 3.2. Mice weresacrificed after 24 hrs, the nasal bone was removed and incubated for 4days with Hela cells in a 24 well plate. If the nasal epithelial cellsare infected, rhinovirus will be released due to the cytopathic effectof the virus and the in vivo infection can then be determined andquantified by measuring cell death of Hela cells as a bioassay. To thisend, dead cells were collected, adherent cells were trypsinized, thefractions were combined, washed twice in H/S (20 mM HEPES, 132 mM NaCl,5 mM KCl, 1 mM CaCl₂), 0.7 mM MgCl₂, 0.8 mM MgSO₄, pH 7.4), stained withTrypan Blue (0.2% final concentration) and dead cells were counted inaliquots of 500 cells as above. Results are illustrated in FIG. 23 andgiven as the percentage of dead cells±SD from 4 independent experimentseach; *p<0.05, ANOVA.

The results show that intranasal application of RV2 into wild-type miceresulted in an infection with RV2, which was prevented by prophylacticapplication of sphingosine or by application of sphingosine 1, 4 or 8hrs after the infection. OGP was without effect on the infection.

3.5

Sphingosine (SPH) that was immobilized to agarose beads (EchelonBiosciences), or control beads (Ctrl) were incubated with 10⁴ PFU ofrhinovirus 2 or 14 (RV2 or RV14) for 4 hrs at 4° C. in a volume of 400μL PBS. The beads were then centrifuged for 2 min at 14 000 rpm in acentrifuge, the supernatant was completely removed. The supernatant wasthen incubated for 4 days with Hela cells in 24 well plates (grown to70% confluency) to determine the remaining viral titer in thesupernatant and, thus, to determine binding of the virus to sphingosineand thereby depletion of the virus from the supernatant. The supernatantwas added to Hela cells and as readout for remaining virus the number ofdead Hela cells in aliquots of 500 cells after 4 days of culture wasdetermined. FIG. 24 illustrates the percentage of dead cells±SD from 5independent experiments each; *p<0.05, ANOVA. The results show thatsphingosine-coupled agarose beads efficiently bound RV2 or RV14 andalmost completely depleted the supernatant from any virus.

As a complement to the above study, RV1b (obtained from ATCC) wereimmobilized to agarose beads and tested for binding of sphingosine(SPH). To this end, we incubated 1 μg/ml anti-RV1b antibodies with 10⁶CFU RV1b for 1 h at 4° C. in H/S, added 50 μL agarose protein A/G beads(Santa Cruz Inc.), incubated for an additional 1 h at 4° C. and washedthe immobilized precipitates 5-times in H/S. We resuspended the beads in500 μL PBS (pH 7.0) containing 250 μM sphingosine. Samples wereincubated for 4 hrs at 4° C., pelleted and aliquots of the supernatantswere added to 50 mM HEPES (pH 7.4), 250 mM NaCl, 30 mM MgCl₂, 0.001units sphingosine kinase (R&D), 1 mM ATP and 10 μCi [³²P]γATP. Thekinase reaction was performed for 1 hr at 30° C., stopped by addition of20 μl 1N HCl, 800 μl CHCl₃/CH₃OH/1N HCl (100:200:1, v/v/v), 240 μl CHCl₃and 2 M KCl. Phases were separated, the lower phase was collected,dried, dissolved in 20 μL of CHCl₃:CH₃OH (1:1, v/v) and separated onSilica G60 thin layer chromatography (TLC) plates usingCHCl₃/CH₃OH/acetic acid/H₂O (90:90:15:5, v/v/v/v). The TLC plates wereexposed to radiography films, spots were removed from the plates, andthe incorporation of [³²P] into sphingosine measured by liquidscintillation counting. Sphingosine was determined using a standardcurve of C18-SPH. Given is the mean of the remaining sphingosineconcentration in the supernatant±SD from 5 independent experiments each;***p<0.001, ANOVA.

To test whether binding of sphingosine (SPH) to RV2 or RV14 depends onthe pH and, thus, presumably on protonation of the NH₂ group insphingosine, sphingosine-agarose beads or control (Ctrl) beads wereincubated with 10⁴ PFU RV2 or RV14 for 4 hrs at 4° C. in a volume of 400μL 150 mM sodium acetate adjusted to pH 6.0, pH 7.0 or pH 8.0. Studieswhere then performed as in the preceding two paragraphs and determinedthe number of virus in the supernatant by a bioassay using thecytopathic effect of the virus on Hela cells as readout. Given is themean of the percentage of dead Hela cells±SD from 5 independentexperiments each; ***p<0.001, ANOVA. The results as illustrated in FIG.26 demonstrate that sphingosine-coupled agarose beads bind virus and,thus, deplete the supernatant from virus at pH 6.0 and pH 7.0, whilebinding of sphingosine to RV2 or RV14 is abrogated at pH 8.0. Thissuggests that protonation of the NH₂ group in sphingosine determinesdirect binding of sphingosine to the viruses. Control beads did not bindrhinovirus.

3.6

10⁴ PFU of Rhinovirus 2 or 14 (RV2, RV14) were incubated with freshlyisolated human epithelial cells for 24 hrs in the presence of 10 μM or20 μM sphingosine (SPH) or the corresponding concentration of thesolvent octylglucopyranoside (OGP), the cells were pelleted,supernatants completely removed, the cell pellets resuspended in PBS andadded to Hela cells. Cells were incubated for 3 days and cytotoxicity asmeasurement for virus titers was determined. Shown is the mean±SD, n=4,*p<0.001 ANOVA. As shown in FIG. 27, the presence of sphingosineabrogates infection of human epithelial cells by both RV2 and RV14.

4. The Effect of Sphingosine on SARS-CoV-2 Infections

To investigate whether exogenous sphingosine prevents the infection ofepithelial cells with SARS-CoV-2, Vero epithelial cells were treatedwith 0.25 μM to 5 μM sphingosine (Avanti Polar Lipids, CAS #: 123-78-4,cat. No. 860490) for 30 min and then infected with pp-VSV-SARS-CoV-2spike. It has previously been shown that pp-VSV-SARS-CoV-2 spikeparticles accurately reflect key aspects of the entry of coronavirusinto host cells (Hoffmann, M., et al., Cell, 2020, 181, 271-80), inparticular they bind to ACE2 for infectious entry, which was inhibitedby anti-ACE2 antibodies.

Pseudotyped viral particles were based on a replication-deficientvesicular stomatitis virus as previously described (Kleine-Weber, H., etal., J. Virol., 2019; 93: e01381-18.). The particles encode for enhancedgreen fluorescent protein (eGFP) and firefly luciferase instead ofparental VSV-G, VSV*AG-FLuc (Berger Rentsch, M., and Zimmer, G., PLoSOne, 2011; 6: e25858). HEK-293T cells were transiently transfected for24 h using the calcium-phosphate method to express either SARS-2-Spikeor VSV-G. Cells were then inoculated with VSV-G-trans-complementedVSV*AG-FLuc for 1 h at 37° C. and 5% CO₂. The inoculum was then removed,cells were washed with PBS and cultured in fresh medium at 37° C. and 5%CO₂ for 16 h. In case of SARS-2-Spike-expressing cells, the culturemedium was supplemented with anti-VSV-G antibody (I1, mouse hybridomasupernatant from CRL-2700; ATCC) to inactivate residualVSV-G-trans-complemented VSV*AG-FLuc. Culture supernatants were finallyharvested and cellular debris was pelleted by centrifugation (4000×g, 4°C., 10 min). The clarified supernatants were used for the experiments.

Vero cells (ATCC©CCL-81™; monkey kidney epithelial cells) were culturedin Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 mM HEPES(pH 7.4; Carl Roth GmbH, Karlsruhe, Germany), 2 mM L-glutamine, 1 mMsodium pyruvate, 100 μM nonessential amino acids, 100 U/mL penicillin,100 μg/mL streptomycin (all from Invitrogen), and 10% FCS (PAALaboratories GmbH, Coelbe, Germany). The cells were grown tosub-confluency for 24 h on glass cover slips in a 24-well plate for 24 hprior to the experiments. Cells were then washed once with H/S andincubated with 0.25 μM, 0.5 μM, 1 μM, 2 μM or 5 μM sphingosine or leftuntreated for 30 min in H/S. The supernatant was removed and cells wereinfected with pp-VSV-SARS-CoV-2 spike in the presence of the sameconcentration of sphingosine as used during the pre-incubation period.Infection was terminated after 60 min, the medium removed and the cellscultured for additional 24 h in DMEM medium supplemented as above toallow expression of eGFP. The medium was removed; cells were washed oncein H/S, fixed in 1% PFA buffered with PBS (pH 7.3) for 10 min, washed,embedded in Mowiol, and analyzed with a Leica TCS-SP5 confocalmicroscope equipped with a 40× lens and Leica LCS software version 2.61.eGFP-positive cells were counted in 2000 cells/sample in randomly chosenmicroscopic fields.). To control for specificity of sphingosine the sametests were repeated using other lipids, i.e. phosphatidylcholine,phosphatidyl-serine, phosphatidylethanolamine, sphingomyelin,C16-ceramide, sphingosine 1-phosphate, lactosyl-ceramide, cardiolipin,and octyl-glucopyranoside.

As illustrated in FIG. 28A concentrations as low 0.25 μM sphingosineshowed a marked effect and 1 μM sphingosine reduced the infection ofVero epithelial cells with pp-VSV-SARS-CoV-2 by more than 90%. None ofcontrol lipids inhibited the infection of Vero cells withpp-VSV-SARS-CoV-2 spike (FIG. 28B). No toxic effects were observed bythe sphingosine administration as illustrated by FITC-Annexin V andTUNEL studies (FIGS. 28C and D), as well as by actin staining usingFITC-phalloidin staining and viewed by confocal microscopy (data notshown).

Further studies were performed in human nasal epithelial cells. Briefly,human nasal epithelial cells were obtained from healthy volunteers bynasal brushings with a small brush. Cells were suspended immediately inHEPES/Saline (H/S; 132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mMCaCl₂), 0.7 mM MgCl₂, 0.8 mM MgSO₄), washed once and resuspended in H/S.Cells were treated with 0.25 μM, 0.5 μM, 1 μM, 2 μM or 5 μM sphingosineor left untreated for 30 min, pelleted and resuspended in MEMsupplemented with 10% FCS containing pp-VSV-SARS-CoV-2 spike and thesame concentration of sphingosine as used in the pre-incubation period.Cells were then infected for 60 min, washed once in H/S and cultured for24 h in MEM supplemented with 10% FCS to allow expression of the eGFPencoded by the particles. Infection was analyzed on a Leica TCS-SP5confocal microscope by counting the percentage of eGFP-positiveepithelial cells in at least 500 epithelial cells per sample in randomlychosen microscopic fields. The local ethics committee approved theexperiments under the number 20-9348-BO.

In human nasal epithelial cells, 1 or 2 μM sphingosine also preventedinfection (FIG. 29A, B). Infection was quantified by counting eGFPpositive cells (FIG. 29A). FIG. 29B shows a representative example ofcells infected with pp-VSV-SARS-CoV-2 spike in the absence (FIG. 29B,left) or presence of 2 μM sphingosine (FIG. 29B right). The greenfluorescence of infected cells is easily detected over the background(autofluorescence of the cells). Sphingosine had no toxic effects onnasal epithelial cells by studies performed as per the above Vero cellstudies (FIG. 29C, D).

To determine if sphingosine (SPH) bound to ACE2, the receptor forSARS-CoV-2 in cells, the interaction of cellular ACE2 or Fc-ACE2 (Abcam,#ab273687) with sphingosine at a concentration of 2 μM was studied. Asillustrated in FIG. 30A-B, cellular or recombinant ACE2 binds tosphingosine beads (FIG. 30A), while binding of the spike protein tosphingosine beads was not detected (not shown). Addition of solublesphingosine prevented binding of ACE2 to immobilized sphingosinesupporting the notion of a specific interaction between ACE2 andsphingosine (FIG. 30A, left panel). Likewise, incubation of immobilizedrecombinant ACE2 protein with sphingosine revealed a binding ofsphingosine to ACE2 (FIG. 30A, right panel).

In addition it was studied whether sphingosine interferes with thebinding of viral spike protein with ACE2. To this end, immobilizedrecombinant Fc-ACE2 was incubated with either 1 μM or 2 μM sphingosineor left untreated, washed and then the recombinant receptor bindingdomain of spike S1 was added. Pre-incubation of ACE2 with sphingosineprevented binding of recombinant receptor binding domain of spike S1 toACE2 (FIG. 30B). Similar to above, phosphatidylcholine,phosphatidyl-ethanolamine, sphingomyelin, ceramide, sphingosine1-phosphate or cardiolipin showed no interation with the beads. Theresults show that sphingosine selectively couples to beads with boundrecombinant ACE2 (FIG. 30C).

The possibility of sphingosine interfering with the binding of viralspike protein with ACE2 was further studied. To this end, immobilizedrecombinant Fc-ACE2 was incubated with 2 μM sphingosine or leftuntreated, washed and then the recombinant receptor-binding domain ofspike S1 was added. Pre-incubation of ACE2 with sphingosine reducedbinding of the recombinant RBD of spike S1 to ACE2 (FIG. 30D). Controllipids did not affect binding of binding of recombinant receptor-bindingdomain of spike S1 to immobilized recombinant ACE2 (FIG. 30E).

A recombinant protein of the receptor-binding domain of the viral spikeprotein (His tagged) was immobilized on Ni²⁺-agarose followed by theaddition recombinant Fc-ACE2 in the presence or absence of 1 μM or 2 μMsphingosine. The results show that sphingosine abolished the interactionof the receptor-binding domain of spike protein with ACE2 (FIG. 30F).Once again, control lipids did not affect the interaction of immobilizedrecombinant Spike-protein with ACE2 (FIG. 30G).

Methods to 0180 and 0183: 2 μg Fc-ACE2 was immobilized on 30 μL proteinA/G agarose beads in 500 μL H/S+A/L at 4° C. for 120 min in the presenceor absence of 1 μM or 2 μM sphingosine. Controls were protein A/Gagarose beads incubated with 1 μM or 2 μM sphingosine or left untreated.Samples were then washed 3-times in H/S+A/L, resuspended in 500 μLH/S+A/L and incubated with 2 μg recombinant receptor binding domain ofspike (RBD-spike, Sino Biologicals MA FE2702B, #40150-R007) for 60 minat 4° C. Samples were washed 6 times in H/S+A/L, eluted in 1×SDS-samplebuffer, boiled for 5 min at 94° C. and centrifuged at 20 800×g for 5 minat 4° C. The supernatants were separated on 10% SDS-PAGE and analyzedfor binding of RBD-spike by western blotting using anti-SARS-CoV-2-spikeS1 antibodies (1:1000, monoclonal rabbit MA14FE2702-B antibody, SinoBiological #40150-R⁰⁰⁷) followed by AP-coupled anti-rabbit antibodies(1:50 000, Abcam #ab97048) as above. In an additional approach to provethat sphingosine blocks binding of viral spike to human ACE2, weimmobilized 2 μg RBD-spike, which is a His-tagged protein, on Ni-agarose(ThermoFisher #89964) in 500 μL H/S+A/L and added 2 μg recombinantFc-ACE2 in the presence or absence of 1 μM or 2 μM sphingosine. Sampleswere incubated for 60 min, washed 6-times in H/S+A/L, eluted in1×SDS-sample buffer and analyzed by western blotting for ACE2 as aboveusing anti-ACE2 antibodies.

It was further analyzed whether sphingosine also interferes with thebinding of pp-VSV-SARS-CoV-2 to recombinant ACE2. The studies revealedthat 1 or 2 μM sphingosine abrogated binding of intact pp-VSV-SARS-CoV-2to recombinant ACE2 resulting in effective pseudo-infection of Verocells with the remaining pp-VSV-SARS-CoV-2 particles (FIG. 31A). Here,Fc-ACE2 (2 μg) was immobilized on 30 μL protein A/G agarose for 60 minin 500 μL H/S+A/L at 4° C. The complexes were washed twice in H/S+A/L, 2μg sphingosine were added or the complexes were left untreated. Sampleswere incubated at 4° C. for 60 min. Complexes were washed twice inH/S+A/L and 50 μL pp-VSV-SARS-CoV-2-spike were added. Samples wereincubated for 60 min at 4° C., pelleted by centrifugation for 2 min at20 800×g and the supernatants were used to infect Vero cells as ameasurement how much functional virus was depleted from the supernatantas a consequence of binding to immobilized Fc-ACE2.

Adhesion studies were performed of pp-VSV-SARS-CoV-2 on Vero cells inthe presence or absence of sphingosine. Vero cells were infected withpp-VSV-SARS-CoV-2 for 20 minutes in the presence or absence ofsphingosine, phosphatidylcholine, sphingomyelin, C16-ceramide,sphingosine 1-phosphate, lactosyl-ceramide or cardiolipin, and thenstained with anti-spike S1 antibodies. Virus binding to the cells wasdetermined by flow cytometry. As illustrated in FIG. 31B, sphingosineprevented binding of pp-VSV-SARS-CoV-2 to the cell surface. None of theother lipids had an effect on the interaction of the virus with thecells demonstrating the specificity of sphingosine for the prevention ofpp-VSV-SARS-CoV-2 to the cell surface.

Overall, the results clearly demonstrate that exogenous sphingosineprevents infection of cells, including freshly isolated human nasalepithelial cells with pp-VSV-SARS-CoV-2.

The foregoing description of particular aspect(s) is merely exemplary innature and is in no way intended to limit the scope of the invention,its application, or uses, which may, of course, vary. The invention isdescribed with relation to the non-limiting definitions and terminologyincluded herein. These definitions and terminology are not designed tofunction as a limitation on the scope or practice of the invention butare presented for illustrative and descriptive purposes only. While theprocesses or compositions are described as an order of individual stepsor using specific materials, it is appreciated that steps or materialsmay be interchangeable such that the description of the invention mayinclude multiple parts or steps arranged in many ways as is readilyappreciated by one of skill in the art.

It will be understood that, although the terms “first,” “second,”“third” etc. may be used herein to describe various elements,components, regions, layers, and/or sections, these elements,components, regions, layers, and/or sections should not be limited bythese terms. These terms are only used to distinguish one element,component, region, layer, or section from another element, component,region, layer, or section. Thus, “a first element,” “component,”“region,” “layer,” or “section” discussed below could be termed a second(or other) element, component, region, layer, or section withoutdeparting from the teachings herein.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms, including “at least one,” unless the content clearly indicatesotherwise. “Or” means “and/or.” As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. It will be further understood that the terms “comprises”and/or “comprising,” or “includes” and/or “including” when used in thisspecification, specify the presence of stated features, regions,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,regions, integers, steps, operations, elements, components, and/orgroups thereof. The term “or a combination thereof” means a combinationincluding at least one of the foregoing elements.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure belongs. It willbe further understood that terms such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified.

Patents, publications, and applications mentioned in the specificationare indicative of the levels of those skilled in the art to which theinvention pertains. These patents, publications, and applications areincorporated herein by reference to the same extent as if eachindividual patent, publication, or application was specifically andindividually incorporated herein by reference.

The foregoing description is illustrative of particular aspects of theinvention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

The invention claimed is:
 1. A process of treating or preventinginfection and/or viral infectious disease by SARS-CoV-2 in a subjectcomprising administering to a subject in need treatment of a viralrespiratory infection or at risk of exposure to SARS-CoV-2, acomposition comprising: a sphingoid compound of formula I:

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical with a substituent of amine,a quaternary ammonium optionally including 3 hydrogens, or a sugarradical, R⁴ and R⁵ independently of one another mean a hydrogen atom,oxygen atom, a hydroxyl group, a quaternary ammonium, or an amine, R⁶ isan ammonium, an ammonium comprising three hydrogens, or

R² and R³ independently of one another mean a hydrogen atom or an alkylradical, n means an integer of from 2 to 50, and

means a double bond or single bond, and/or salts thereof and/or opticalisomers, and/or diastereomers and/or epimers thereof and/or mixturesthereof; thereby treating or preventing a viral infection in thesubject.
 2. The process of claim 1 wherein the composition comprisessphingosine.
 3. The process of claim 1 wherein the compositioncomprises: a sphingoid compound of formula I-a

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical, an ammonium, an ammoniumincluding 3 hydrogens, or a sugar radical, R² and R³ independently ofone another mean a hydrogen atom or an alkyl radical, R⁴ and R⁵independently of one another mean a hydrogen atom, an ammonium, anammonium including 3 hydrogens, or a hydroxyl group, n means an integerof from 2 to 50, and

means a double bond or single bond, and/or salts thereof and/or opticalisomers, optionally enantiomers and/or diastereomers and/or epimers,thereof and/or mixtures thereof.
 4. The process of claim 1 wherein thecomposition comprises: a sphingoid compound of formula I-b

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical, an ammonium, an ammoniumincluding 3 hydrogens, or a sugar radical, R² and R³ independently ofone another mean a hydrogen atom or an alkyl radical, R⁴ and R⁵independently of one another mean a hydrogen atom, an ammonium, anammonium including 3 hydrogens, or a hydroxyl group, and n means aninteger of from 2 to
 50. 5. The process of claim 1 wherein thecomposition comprises: a sphingoid compound of formula I-c

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical, an ammonium, an ammoniumincluding 3 hydrogens, or a sugar radical, R² and R³ independently ofone another mean a hydrogen atom or an alkyl radical, R⁴ and R⁵independently of one another mean a hydrogen atom, oxygen atom, anammonium, an ammonium including 3 hydrogens, or a hydroxyl group, and nmeans an integer of from 2 to
 50. 6. The process of claim 1 wherein thecomposition comprises: a sphingoid compound of formula II

where n means an integer of from 2 to 50, and

means a double bond or single bond.
 7. The process of claim 1 whereinthe composition comprises: a sphingoid compound of formula II-a₀:

where n means an integer of from 2 to 50; or a sphingoid compound offormula II-a₀*:

where n means an integer of from 2 to
 50. 8. The process of claim 1wherein the sphingoid compound comprises sphingosine,dihydrosphingosine, phytosphingosine, dehydrophytosphingosine, saltsthereof, hydrates thereof, solvates thereof, polymorphs thereof, opticalisomers, or mixtures of at least two thereof.
 9. The process of claim 1wherein the composition is prepared for administration by intravenous,intra-arterial, cutaneous, subcutaneous, percutaneous, intramuscular,inhalational, intravaginal, oral, nasal, or conjunctival administration.10. The process of claim 1 wherein the administering is intravenous,nasal, pharyngeal, tracheal and pulmonary administration, oradministration is by injection, spray, dry powder or inhalation.
 11. Theprocess of claim 1 wherein the subject is a human, the administration isnasal, and the composition comprises sphingosine.
 12. The process ofclaim 1 wherein said process treats SARS-CoV-2 virus infection in saidsubject.
 13. The process of claim 1 wherein said compound comprises: asphingoid compound of formula I-a

where R¹ means a hydrogen atom, a methyl radical or —CH₂OR^(a), whereR^(a) is a hydrogen atom, an alkyl radical of 1-4 carbons, an ammonium,an ammonium including 3 hydrogens, or a sugar radical, R² and R³independently of one another mean a hydrogen atom or a methyl radical,R⁴ and R⁵ independently of one another mean a hydrogen atom, anammonium, an ammonium including 3 hydrogens, or a hydroxyl group, wheren means an integer of from 2 to 50, and

means a double bond or single bond, and/or salts thereof and/or opticalisomers, enantiomers and/or diastereomers and/or epimers, thereof and/ormixtures thereof.
 14. The process of claim 12 wherein saidadministration is nasal.
 15. The process of claim 13 wherein saidadministration is nasal.
 16. The process of claim 1, wherein n is 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.